This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding starch R1 phosphorylation proteins in plants and seeds.
Starch is a mixture of two polysaccharides, amylose and amylopectin. Amylose is an unbranched chain of up to several thousand xcex1-D-glucopyranose units linked by xcex1-1,4 glycosidic bonds. Amylopectin is a highly branched molecule made up of up to 50,000 xcex1-D-glucopyranose residues linked by xcex1-1,4 and xcex1-1,6 glycosidic bonds. Approximately 5% of the glycosidic linkages in amylopectin are xcex1-1,6 bonds, which leads to the branched structure of the polymer.
Amylose and amylopectin molecules are organized into granules that are stored in plastids. The starch granules produced by most plants are 15-30% amylose and 70-85% amylopectin. The ratio of amylose to amylopectin and the degree of branching of amylopectin affects the physical and functional properties of the starch. Functional properties, such as viscosity and stability of a gelatinized starch determine the usefulness and hence the value of starches in food and industrial applications.
The R1 protein of potato appears to be a granule associated enzyme that is involved in starch phosphorylation (Lorberth, R. et al. (1998) Nature Biotechnology 16:473-477). Nucleic acid fragments encoding starch R1 phosphorylation proteins have been isolated from other species, including rice (PCT International Application No. PCT/EP99/08506) and corn (Patent Application No. DE19653176-A1).
R1 activity has been associated with starch degradation in potato tubers. Studies have shown that inhibition of R1 activity leads to the synthesis of modified starch that is not degraded by enzymes present in potato tissue. If changes in starch degradation are a direct consequence of changes in the degree of phosphorylation this suggests that starch phosphorylation is an important modification that promotes degradation.
Accordingly, the availability of nucleic acid sequences encoding all or a portion of R1 proteins in other plants would facilitate studies to better understand starch degradation and provide genetic tools for the manipulation of starch modification, biosynthesis and metabolism in plant cells.
The present invention concerns an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 50 or 100 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:6 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:14 have at least 90% or 95% identity based on the Clustal alignment method, (c) a third nucleotide sequence encoding a third polypeptide comprising at least 150 amino acids, wherein the amino acid sequence of the third polypeptide and the amino acid sequence of SEQ ID NO:2 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (d) a fourth nucleotide sequence encoding a fourth polypeptide comprising at least 150 amino acids, wherein the amino acid sequence of the fourth polypeptide and the amino acid sequence of SEQ ID NO:10 have at least 85%, 90%, or 95% identity based on the Clustal alignment method, (e) a fifth nucleotide sequence encoding a fifth polypeptide comprising at least 350 amino acids, wherein the amino acid sequence of the fifth polypeptide and the amino acid sequence of SEQ ID NO:12 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (f) a sixth nucleotide sequence encoding a sixth polypeptide comprising at least 600 amino acids, wherein the amino acid sequence of the sixth polypeptide and the amino acid sequence of SEQ ID NO:20 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (g) a seventh nucleotide sequence encoding a seventh polypeptide comprising at least 1337 amino acids, wherein the amino acid sequence of the seventh polypeptide and the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:18 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, or (h) the complement of the first, second, third, fourth, fifth, sixth, or seventh nucleotide sequence, wherein the complement and the first, second, third, fourth, fifth, sixth, or seventh nucleotide sequence contain the same number of nucleotides and are 100% complementary. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:6, the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:14, the third polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, the fourth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:10, the fifth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:12, the sixth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:20, and the seventh polypeptide preferably comprises the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:18. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:5, the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:13, the third nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, the fourth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:9, the fifth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:11, the sixth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:19, and the seventh nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:15 or SEQ ID NO:17. The first, second, third, fourth, fifth, sixth, and seventh polypeptides preferably are starch R1 phosphorylation proteins.
In a second embodiment, the present invention relates to a chimeric gene comprising any of the isolated polynucleotides of the present invention operably linked to a regulatory sequence.
In a third embodiment, the present invention relates to a vector comprising any of the isolated polynucleotides of the present invention.
In a fourth embodiment, the present invention relates to an isolated polynucleotide fragment comprising a nucleotide sequence comprised by any of the polynucleotides of the present invention, wherein the nucleotide sequence contains at least 30, 40, or 60 nucleotides.
In a fifth embodiment, the present invention relates to an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 50 or 100 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:6 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (b) a second amino acid sequence comprising at least 100 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:14 have at least 90% or 95% identity based on the Clustal alignment method, (c) a third amino acid sequence comprising at least 150 amino acids, wherein the third amino acid sequence and the amino acid sequence of SEQ ID NO:2 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (d) a fourth amino acid sequence comprising at least 150 amino acids, wherein the fourth amino acid sequence and the amino acid sequence of SEQ ID NO:10 have at least 85%, 90%, or 95% identity based on the Clustal alignment method, (e) a fifth amino acid sequence comprising at least 350 amino acids, wherein the fifth amino acid sequence and the amino acid sequence of SEQ ID NO:12 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (f) a sixth amino acid sequence comprising at least 600 amino acids, wherein the sixth amino acid sequence and the amino acid sequence of SEQ ID NO:20 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, or (g) a seventh amino acid sequence comprising at least 1337 amino acids, wherein the seventh amino acid sequence and the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:18 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method. The first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:6, the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:14, the third amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:2, the fourth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:10, the fifth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:12, the sixth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:20, and the seventh amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:18. The polypeptide preferably is a starch R1 phosphorylation protein.
In a sixth embodiment, the present invention relates to a method for transforming a cell comprising introducing any of the isolated polynucleotides of the present invention into a cell, and the cell transformed by this method. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.
In a seventh embodiment, the present invention relates to a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the chimeric genes of the present invention.
In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a starch R1 phosphorylation protein or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level of the starch R1 phosphorylation protein or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the starch R1 phosphorylation protein or enzyme activity in the host cell containing the isolated polynucleotide with the level of the starch R1 phosphorylation protein or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a starch R1 phosphorylation protein, preferably a plant starch R1 phosphorylation protein, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a starch R1 phosphorylation protein amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a starch R1 phosphorylation protein comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In an eleventh embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the starch R1 phosphorylation protein polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a twelfth embodiment, this invention relates to a method of altering the level of expression of a starch R1 phosphorylation protein in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the starch R1 phosphorylation protein in the transformed host cell.